Previous studies have shown that a mutant (designated C4) of Streptococcus mutans 6715 wild type (WT) is defective in glucosyltransferase (GTF)-synthesized insoluble glucan and is avirulent in gnotobiotic rats. This study investigated the factors which would render this mutant virulent in gnotobiotic rats. Microbial analysis of plaque from gnotobiotic rats (45 days old) infected with a mixture of C4 and virulent S. mutans PS-14 (approximately 15,000 C4 organisms to each S. mutans PS-14) yielded higher numbers of C4 organisms than S. mutans PS-14. These animals exhibited significantly lower caries scores than did gnotobiotic rats (age, 45 days) monoassociated with S. mutans PS-14. Similar mixed infection studies using C4 and an avirulent, aggregation-defective mutant of S. mutans 6715 WT (designated UAB 165) which exhibits GTF activity similar to that of the parent strain resulted in plaque consisting almost exclusively of UAB 165 and low caries activity. However, high levels of both C4 and UAB 165 in plaque and high caries activity were observed in gnotobiotic rats infected at weaning with C4 followed by UAB 165 3 days later. When dried S. mutans 6715 WT culture supernatant containing GTF activity was mixed with diet provided rats monoassociated with C4, significant caries activity was observed. Insoluble glucan supplemented in diet did not restore C4 to virulence; however, admixture of suboptimal GTF-rich supernatant with insoluble glucan and C4 resulted in high caries activity in gnotobiotic rats. These results suggest that in vivo restoration of pathogenesis of a GTF-defective mutant of S. mutans can be achieved either by complementation with a mutant defective in aggregation properties or by providing exogenous GTF and glucan from the parent S. mutans 6715 WT.